Vascular endothelial cells (ECs) are constantly subjected to cyclic stretch due to the pulsatile pressure. In straight, unbranched arteries, ECs are elongated with their major axes oriented with that of the arterial vessel, i.e. perpendicular to the direction of stretch. Fibers aligned perpendicular to stretch bear less tension than when they are aligned parallel to stretch. Thus, perpendicular orientation of the stress fibers serves to reduce the stretch- induced tension and thus minimize the intracellular mechanical energy produced by uniaxial stretch. We propose a mechanical and molecular feedback control model that leads to energy minimization by EC stress fiber remodeling. Our preliminary experiments have shown that an increase of Rho activity in ECs enhances the perpendicular orientation of stress fibers to stretch and that the inhibitionof Rho activity causes the stress fibers to be oriented parallel to the direction of stretch. These results led us to propose that Rho GTPase has a central role in regulating the stretch-generation of intracellular mechanical energy through the control of stress fiber re- organization. We propose to test our model with studies listed under the following specific aims: (1) To determine the change in intracellular energy from measured cell viscoelastic properties and strain rate during mechanical stretch: Magnetic twisting cytometry and intracellular particle displacement will be used to determine cell viscoelastic properties which, together with the measurement of stretch-induced strain rate, will allow the computation of intracellular energy. (2) To establish the effects of different modes of mechanical stretch on stress fiber orientation and intracellular energy: We will measure the intracellular energy resulting from various modes of stretch and track changes in intracellular energy due to stress fiber remodeling and modulation of myosin light chain activity. (3) To elucidate the role of Rho GTPase pathway in stretch-induced changes in stress fiber orientation and intracellular energy: The time courses of activation of the Rho/ROCK/MLC pathway, as well as the effects of modulating their activities, will be related to stress fiber remodeling and intracellular energy. (4) To determine the effects of different modes of stretch on mechanotransduction and EC proliferation/apoptosis: The relevance of stretch-induced intracellular energy on the activation of MAPKs and their role in cell fate will be tested. (5) To test the validity of the in vitro results in an ex vivo artery system: The effects of different modes of stretch on stress fiber remodeling, intracellular signaling and cell fate will be measured in the intact endothelium of excised arteries. The results generated from these proposed studies will provide insight into the cellular adaptation mechanisms and homeostasis of biological functions in responses to stretch.